Gel having biosubstance fixed thereto and microarray utilizing the gel

ABSTRACT

The present invention provides a biological substance-immobilized gel which comprises a gel containing 2%-7% by mass of N,N-dimethylacrylamide and a biological substance immobilized on and/or in the gel.

TECHNICAL FIELD

The present invention relates to a biological substance-immobilized geland a biological substance-immobilized gel microarray using the same.The microarray is used for analysis of gene expression, etc.

BACKGROUND ART

The decoding of human genome is now progressing and helping clarifycausal relations between various diseases or diatheses and specific genesequences. For example, such a gene analysis is intended to use forpredicting the onset of diseases, side effects of drugs, etc.

A means conventionally used for gene analysis is gel-basedelectrophoresis. In recent years, capillary gel electrophoresis has beendeveloped with the aim of separating and analyzing trace amounts ofbiological samples in a short time. Capillary gel electrophoresis usesglass capillaries filled with a hydrogel such as acrylamide.

In addition, microarrays carrying multiple capture probes for DNA orprotein detection (i.e., probes capable of capturing target DNA orprotein molecules and the like through hybridization or binding to theDNA or protein molecules to be detected) are employed as useful toolsfor detecting mutations and expression levels of many genes all at once.Such microarrays are also known to have a large number of variationswhich are constructed using a gel. Among them, microarrays known to usea gel for immobilization of capture probes include, for example, thosehaving multiple slots or holes on a substrate (e.g., a resin board), inwhich the slots or holes are filled with a DNA-containing gel (see JP2000-60554 A), as well as those having gel spots containing DNA or othersubstances on a flat substrate (see U.S. Pat. No. 5,770,721). Also, someof the inventors of the present invention have developed a microarraythat is obtained by creating a hollow fiber alignment comprising hollowfibers whose hollow space is filled with a capture probe-containing geland then cutting the alignment in a direction intersecting with itsfiber axis. This microarray has been filed for patent application (seeJP 2000-270877 A, JP 2000-270878 A and JP 2000-270879 A).

These capture probe-immobilized microarrays may be used forhybridization with an analyte to detect specific nucleotide sequences.Detection of hybrids is accomplished by using a known means capable ofspecifically recognizing the hybrids, as exemplified by fluorescencedetection.

However, there has been a problem that when the microarrays afterhybridization are measured for fluorescence intensity in each of theircompartments where capture probes are immobilized, the fluorescenceintensity is higher in the outer regions of the compartments, but lowerin the center regions of the compartments.

DISCLOSURE OF THE INVENTION

The object of the present invention is to obtain gel composition whichensures a uniform distribution of fluorescence intensity in eachcompartment and provides a higher value for total fluorescence intensitysummed over the entire area of each compartment, i.e., higherhybridization efficiency in the detection of a microarray afterhybridization.

As a result of extensive and intensive efforts made to overcome theproblem stated above, the inventors of the present invention have foundthat when capture probes are immobilized on and/or in a gel satisfyingthe following properties, it is possible to ensure a uniformdistribution and increased level of fluorescence intensity in eachcompartment, i.e., higher hybridization efficiency. This finding led tothe completion of the present invention.

Namely, the present invention provides a biologicalsubstance-immobilized gel which comprises a gel containing 2%-7% by massof N,N-dimethylacrylamide and a biological substance immobilized onand/or in the gel. The present invention also provides a biologicalsubstance-immobilized gel which comprises a gel having the followingcomposition and a biological substance immobilized on and/or in the gel:(a) N,N-dimethylacrylamide   2% to 7% by mass (b) cross-linking agent0.1% to 1.5% by mass.

In the above biological substance-immobilized gel, examples of abiological substance include nucleic acids. On the other hand, examplesof a cross-linking agent include multifunctional monomers having atleast two ethylenically unsaturated bonds, as exemplified bymethylenebisacrylamide.

The present invention further provides a method for preparing abiological substance-immobilized gel, which comprises immobilizing abiological substance on and/or in a gel containing 2%-7% by mass ofN,N-dimethylacrylamide. In the present invention, the gel is preferablyobtained by reacting 2%-7% by mass of N,N-dimethylacrylamide in thepresence of 0.1%-1.5% by mass of a cross-linking agent.

The present invention further provides a gel-filled hollow tube whichcomprises a hollow tube whose hollow space is filled with the biologicalsubstance-immobilized gel mentioned above. Examples of a hollow tubeinclude hollow fibers.

The present invention further provides a method for manufacturing abiological substance-immobilized gel microarray, which comprisesallowing a plurality of the above gel-filled hollow tubes to be tied ina bundle and cutting the tube bundle in a direction intersecting withthe longitudinal direction of the tubes.

The present invention further provides a method for manufacturing abiological substance-immobilized gel microarray, which comprises thefollowing steps:

-   (a) allowing a plurality of hollow tubes to be tied in a bundle;-   (b) filling the above biological substance-immobilized gel into the    hollow space of each tube in the resulting tube bundle; and-   (c) cutting the tube bundle in a direction intersecting with the    longitudinal direction of the tubes.

The present invention further provides a biologicalsubstance-immobilized gel microarray which comprises the abovebiological substance-immobilized gel arranged in multiple compartments.In this case, the surface area of each compartment is preferably 10⁻⁶ m²or less. It is also possible to employ a biologicalsubstance-immobilized gel microarray whose compartments are formed byslots or through holes.

The present invention further provides a biologicalsubstance-immobilized gel microarray which is obtained by allowing aplurality of the above gel-filled hollow tubes (e.g., hollow fibers) tobe tied in a bundle and cutting the tube bundle in a directionintersecting with the longitudinal direction of the tubes.

The present invention further provides a method for detecting a targetto be measured (e.g., nucleic acids such as DNA), which comprisesreacting an analyte with the microarray mentioned above and detectingthe target in the analyte. In a case where a target to be measured inthis detection is DNA, it is preferably 100 nucleotides or less inlength.

The present invention will be described in more detail below.

The present invention is directed to a gel comprising a biologicalsubstance immobilized thereon and/or therein (i.e., a biologicalsubstance-immobilized gel), whose composition includesN,N-dimethylacrylamide (2% to 7% by mass).

As used herein, the term “biological substance” is intended to mean abiological material which may be used as a capture probe. Examplesinclude deoxyribonucleic acids (DNA), ribonucleic acids (RNA), proteinsand lipids. These biological substances may be commercially available orderived from living cells, etc.

For example, DNA extraction from living cells may be accomplished, e.g.,by the method of Blin et al. [Nucleic. Acids. Res. 3. 2303 (1976)],while RNA extraction may be accomplished, e.g., by the method ofFavaloro et al. [Methods. Enzymol. 65. 718 (1980)].

DNA used for this purpose is linear or circular plasmid DNA orchromosomal DNA. It is also possible to use DNA fragments cleaved withrestriction enzymes or by chemical treatments, DNA molecules synthesizedin vitro by enzymatic or other processes, or oligonucleotides chemicallysynthesized, etc.

Biological substances prepared by the methods stated above or othertechniques are immobilized on and/or in a gelatinous material(hereinafter referred to as a gel). As used herein, the term“immobilized” is used to mean that a biological substance is retained onand/or in a gel.

The composition of such a gel includes N,N-dimethylacrylamide in anamount of 2% to 7% by mass of the gel, but the following composition ispreferred: (a) N,N-dimethylacrylamide   2% to 7% by mass (b)cross-linking agent 0.1% to 1.5% by mass.

More preferably, the lower limit of the amount of N,N-dimethylacrylamideis 2.5% to 5.0% by mass.

Preferred cross-linking agents are multifunctional monomers having atleast two ethylenically unsaturated bonds. The amount of such across-linking agent is preferably 0.1% to 1.5% by mass of the gel, andmore preferably 0.3% to 0.7% by mass of the gel. Any cross-linking agentcan be used without particular limitations as long as it is among themultifunctional monomers stated above. Examples includemethylenebisacrylamide, divinylbenzene, and polyethylene glycoldi(meth)acrylate.

To prepare such a gel, for example, N,N-dimethylacrylamide and across-linking agent may be mixed and copolymerized in an aqueous medium,or alternatively, N,N-dimethylacrylamide may be polymerized to give aprepolymer, which in turn may be mixed and copolymerized with across-linking agent.

To immobilize biological substances on and/or in the above gel, forexample, biological substances modified to have a terminal vinyl groupmay be added during polymerization and copolymerized with components ofthe gel (see WO 02/62817), or a hydrazine-treated gel may be preparedand reacted with biological substances having an amino group (see JP6-507486 A).

The biological substance-immobilized gel prepared in the presentinvention preferably has a water permeability of 1.0×10⁻⁵m³·m·/m²/hr/MPa or more. The water permeability of the gel is calculatedfrom the amount of water permeating through the gel. A water permeationexperiment is performed as follows and the measured value is defined asthe water permeability.

A gel disk of 1 mm thickness and 20 mm diameter is prepared and overlaidon a support filter (Millipore SMWPO4700). The gel disk is then placedin a filtration holder (ADVANTEC UHP-43K) and the holder is filled withwater. Nitrogen pressure is then applied to the filtration holder and aPE tube of 2 mm diameter is connected to the filtrate outlet. The amountof water permeating through the gel disk is estimated from the timerequired for the front-end of the filtrate to move a given distance (40cm) through the tube, followed by calculation of the water permeability.

In addition, the gel preferably has a shape retention rate of 0.4 ormore, more preferably 0.6 or more. The shape retention rate of the gelis defined as the value measured as follows.

A gel is prepared in a cylindrical container of 13 mm diameter and 4 cmlength. The gel is removed from the container, allowed to stand at 25°C. for 24 hours in an airtight container, and then measured for itsheight The shape retention rate is then calculated by the followingequation:Shape retention rate=height (mm) of the gel after 24 hours/13 mm(initial diameter of the gel)

The thus prepared biological substance-immobilized gel may be used as atool for gene analysis as a gel carrying capture probes.

For example, the above gel may be filled into the hollow space of ahollow tube to prepare a gel-filled hollow tube, which in turn can beused as an analysis tool for genes, etc. It should be noted that thehollow space may be filled with the gel in the same manner as in theproduction of capillary columns used for capillary gel electrophoresis.

The gel of the present invention may also be used as a component of amicroarray. For example, when the above gel carrying capture probesimmobilized thereon and/or therein (hereinafter referred to as animmobilized gel) is arranged on a flat substrate, it is possible tomanufacture a microarray in which the immobilized gel is arranged inmultiple compartments on the flat substrate (see JP 6-507486 A and U.S.Pat. No. 5,770,721). A flat substrate having multiple slots or throughholes may also be used for this purpose. In this case, a biologicalsubstance-containing monomer solution before or immediately afterinitiation of polymerization may be introduced into each compartmentformed by a slot or a through hole, followed by polymerization andcross-linking within each compartment to give a microarray in which abiological substance-immobilized gel is arranged on the substrate (i.e.,a biological substance-immobilized gel microarray) (see JP 2000-60554A).

The type of biological substance to be retained in each compartment mayvary from compartment to compartment. Alternatively, multipleimmobilized gels of the same type may be grouped together and arrangedon a microarray. Likewise, a gel carrying, e.g., a pigment instead of abiological substance may be retained in a compartment(s) to determinethe coordinates of compartments.

The surface area of each compartment is usually 10⁻⁶ m² or less. Thelower limit is not restricted in any way as long as biologicalsubstances can be detected.

In the present invention, examples of hollow tubes include glass tubes,stainless steel tubes, and hollow fibers. In terms of processability andease of handling, hollow fibers are preferred for use. Examples offibers available for use in the present inventions include chemicalfibers such as synthetic fibers, semi-synthetic fibers, regeneratedfibers and inorganic fibers, as well as natural fibers (JP 2000-270878A). Representative examples of synthetic fibers include various types ofpolyamide-type fibers such as Nylon 6, Nylon 66 and aromatic polyamidefibers, various types of polyester-type fibers such as polyethyleneterephthalate, polybutyrene terephthalate, polylactic acid andpolyglycolic acid fibers, various types of acrylic-type fibers such aspolyacrylonitrile fibers, various types of polyolefin-type fibers suchas polyethylene and polypropylene fibers, various types of polyvinylalcohol-type fibers, various types of polyvinylidene chloride-typefibers, polyvinyl chloride-type fibers, various types ofpolyurethane-type fibers, phenol-type fibers, fluoro-type fibers such aspolyvinylidene fluoride and poly(tetrafluoroethylene), polyalkyleneparahydroxybenzoate-type fibers, as well as fibers formed using(meth)acrylic-type resins such as polymethylmethacrylate.

Representative examples of semi-synthetic fibers include various typesof cellulose-type derivative-type fibers originated from diacetate,triacetate, chitin, chitosan and the like, as well as various types ofprotein-type fibers called promix. Representative examples ofregenerated fibers include various types of regenerated cellulose fibers(e.g., rayon, cupra, polynosic) which are obtained by viscose orcuprammonium process or by organic solvent process.

Representative examples of inorganic fibers include glass fibers andcarbon fibers. Representative examples of natural fibers includevegetable fibers such as cotton, linen, ramie and jute, animal fiberssuch as sheep wool and silk, as well as mineral fibers such as asbestos.

Hollow fibers other than natural fibers may be produced in a knownmanner using special nozzles. The melt spinning technique is preferredfor polyamides, polyesters, polyolefins and the like, which can use ahorseshoe- or C-shaped nozzle, a double-tubed nozzle, etc.

The solvent spinning technique is preferred for spinning syntheticpolymers that are not melt-spinnable and polymers that are used insemi-synthetic fibers or regenerated fibers. As in the case of meltspinning, a double-tubed nozzle is also used in this case to give hollowfibers having a continuous hollow space by spinning the fibers whilefilling an appropriate liquid as a core material into the hollow space.

The hollow tubes thus prepared may each be used as a base unit forsupporting the biological substance-immobilized gel of the presentinvention. In the case of using hollow tubes, microarrays (biologicalsubstance-immobilized gel microarrays) may be manufactured, for example,by allowing a plurality of the above hollow tubes to be tied in abundle, filling the biological substance-immobilized gel into the hollowspace of each hollow tube in the resulting tube bundle, and then cuttingthe tube bundle in a direction intersecting with the longitudinaldirection of the tubes in such a manner as to give cross-sectionalslices (see WO 00/53736). In the present invention, individual hollowtubes may be filled with the gel before being tied in a bundle.

In this case, these hollow tubes may be regularly arranged and bondedwith an adhesive or the like to give, e.g., a tube alignment in whichthe hollow tubes are regularly arranged in both vertical and horizontaldirections. The term “regularly” is used to mean that tubes are arrangedin an orderly manner such that the number of hollow tubes contained in afame of certain size can be the same.

Such a tube alignment may be produced as follows, by way of example.Namely, two perforated plates with a regular arrangement of holes areprovided, and hollow tubes are threaded through the holes in both platessuch that the positions of holes in both perforated plates are matchedwith each other. The space between these perforated plates is thenadjusted. It should be noted that the step of threading hollow tubesthrough the holes and the step of adjusting the space between perforatedplates may be conducted in reverse order. Then, tension is applied tothe hollow tubes and, under this condition, spaces between the hollowtubes (spaces within the tube bundle) are filled with a resin so as tobond the bundle of the tubes, thereby obtaining a tube alignment (JP2001-239594 A).

The tube alignment may be of any shape in cross section. For example,hollow tubes may be regularly arranged to form a square or rectangularcross section, or alternatively, hollow tubes may be concentricallyarranged to form a circular cross section.

In the present invention, the above tube alignment is cut in a directionintersecting with, preferably perpendicular to, the longitudinaldirection (i.e., the axial direction of the hollow tubes) to obtainslices. An example of a cutting method involves cutting slices from thetube alignment using a microtome. The thickness of slices can bearbitrarily adjusted, but it usually ranges from 1 to 5,000 μm,preferably 10 to 2,000 μm.

The slices thus prepared may each be used as a microarray for supportingthe biological substance-immobilized gel.

Biological substances immobilized on and/or in the gel in the microarrayserve as capture probes for nucleic acids or proteins which hybridize orbind to the biological substances (such nucleic acids or proteins beingcalled targets to be measured). Thus, the microarray of the presentinvention can be used as a kit for detecting a target(s) to be measured(e.g., nucleic acids or proteins).

An analyte containing biological substances to be detected (e.g.,nucleic acids such as DNA) is prepared, added to the microarray and thenreacted with biological substances immobilized on and/or in the gel ofthe microarray. For example, DNA targets to be measured arefluorescently labeled and then hybridized with DNA in the microarray.Subsequently, the microarray is washed to remove unreacted DNA, followedby detection of fluorescence intensity. The fluorescence intensity maybe detected using any device (e.g., a commercially available DNAdetector). According to the present invention, the inventive “biologicalsubstance-immobilized gel which comprises a gel containing 2%-7% by massof N,N-dimethylacrylamide and a biological substance immobilized onand/or in the gel” has good reactivity and ensures uniform fluorescenceintensity per compartment of a microarray, thus providing highlysensitive detection results.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 presents photographs showing the results of DNA detection usingthe microarray of the present invention.

FIG. 2 presents photographs showing the results of DNA detection usingthe microarray of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be further described in more detail in thefollowing Examples, which are not intended to limit the scope of theinvention.

EXAMPLE 1

(1) Production of Polymethylmethacrylate (PMMA) Hollow Fibers

An acrylic resin with a mass molecular weight of about 90,000, which wascomposed of methyl methacrylate (MMA) and methyl acrylate (MA) in amonomer ratio of 82:18, was used as a source material and melt-extrudedusing an extruder through a spinning nozzle having a circular outlet,thereby obtaining hollow fibers with an outer diameter of 0.3 mm, aninner diameter of 0.2 mm and a length of 600 mm.

(2) Production of a Hollow Fiber Alignment

Two perforated plates of 0.1 mm thickness were placed one upon another,each of which had 9 holes (diameter: 0.32 mm; center-to-center distance:0.42 mm) arranged in a 3 by 3 array, and 9 hollow fibers prepared abovewere then threaded through the respective holes in these perforatedplates. The space between these two perforated plates was set to 50 mmand the hollow fibers were fixed under tension at two points, 50 mm and100 mm from one end.

A resin raw material was then poured into the space between these twoperforated plates. As a resin, a polyurethane resin adhesive (Nipporan4276/Coronate 4403, Nippon Polyurethane Industry Co., Ltd.) was used,which was supplemented with carbon black in an amount of 2.5% by mass,based on the total weight of this adhesive. The plates were allowed tostand at room temperature for 1 week to cure the resin. The perforatedplates were then removed to give a hollow fiber alignment.

(3) Preparation of an Oligonucleotide having a Terminal Vinyl Group(Vinyl-Terminated Oligonucleotide

Oligonucleotide synthesis was carried out using an automated DNA/RNAsynthesizer (PE Biosystems Model 394). In the final step of synthesis,an amino group [NH₂(CH₂)₆—] was introduced at the 5′-terminus tosynthesize oligonucleotide A (SEQ ID NO: 1) shown below. The sameprocedure was repeated to synthesize oligonucleotide B (SEQ ID NO: 2),except that no amino group was introduced at the 5′-terminus. Aminogroup introduction at the 5′-terminus was accomplished by usingAminoLink II™ (Applied Biosystem).

These oligonucleotides were deprotected and purified in a standardmanner before use. [Oligonucleotide A (SEQ ID NO: 1)] caaccaaccacaactacata cacatac [Oligonucleotide B (SEQ ID NO: 2)] gtcatttagacaactctgca agcgt

Subsequently, oligonucleotide A (500 nmol/ml, 5 μl) and glycidylmethacrylate (0.5 μl) were mixed and reacted at 70° C. for 2 hours.After completion. of the reaction, water was added to a total volume of25 l to give an oligonucleotide (100 nmol/ml) having a terminalmethacrylate group (GMA-denatured oligonucleotide A).

(4) PCR Reaction of a Vinyl-Terminated Oligonucleotide

Saccharomyces cerevisiac JCM7255 was grown in 100 ml YPD medium (20 g/Lglucose, 10 g/L yeast extract, 20 g/L polypeptone, pH 6.0) at 30° C. for1 day, followed by collection of the bacterial cells. The chromosomalDNA was prepared in a routine manner from the collected bacterial cellsand used as a PCR template.

The GMA-denatured oligonucleotide A and oligonucleotide B were dilutedwith sterilized water to 50 μM and 5 μM, respectively. Theseoligonucleotides were used as primers to perform polymerase chainreaction (hereinafter referred to as PCR) with the template preparedabove.

PCR conditions were as described in the specification of Ex-Taq (TakaraShuzo Co., Ltd.) and PCR was performed using a TaKaRa PCR Thermal CyclerPERSONAL. The reaction was repeated for 30 cycles with 100 μl undertemperature conditions of 93° C. for 30 seconds, 65° C. for 30 secondsand 72° C. for 2 minutes. A vinyl-terminated nucleic acid (capture probeA; SEQ ID NO: 3) was amplified by PCR.

(a) Preparation of a Monomer Solution and a Polymerization InitiatorSolution

Polymerization solutions 1 and 2 having the compositions shown in Table1 were prepared. Monomer solution A and a polymerization initiatorsolution were prepared as follows.

[Monomer Solution A]

Dimethylacrylamide (0.45 g) and methylenebisacrylamide (0.05 g) weredissolved in a 50/50 (by mass) mixture of glycerine and pure water togive a total volume of 10 ml.

[Polymerization Initiator Solution]

2,2′-Azobis(2-imidazolin-2-yl)propane) dihydrochloride (1 g) wasdissolved in pure water to give a total volume of 10 ml. TABLE 1Polymerization Polymerization solution 1 solution 2 Monomer solution A1000 μl 1000 μl Polymerization initiator solution  10 μl 10 μl Captureprobe A (100 nmol/ml)   5 μl 0(6) Preparation of Slices

Polymerization solution 1 was filled into the hollow space of threehollow fibers in the center row of the hollow fiber alignment obtainedin (2) above, while polymerization solution 2 was filled into the hollowspace of the other hollow fibers. Polymerization solutions 1 and 2 werefilled. The alignment was transferred to an airtight glass container,inside of which was saturated with water vapor, and then allowed tostand at 55° C. for 1 hour to perform polymerization.

After polymerization, the hollow fiber alignment was repeatedly cutusing a microtome in a direction perpendicular to the longitudinaldirection of the hollow fibers, thereby obtaining slices of about 500 μmthickness.

(7) Hybridization

A hybridization solution was prepared, which was supplemented with 200fmol/ml oligonucleotide C (SEQ ID NO: 4) complementary to a part of thenucleotide sequence of capture probe A (nucleotides 241 to 339 of SEQ IDNO: 3).

Oligonucleotide C was synthesized in the same manner as shown in (3)above using an automated DNA synthesizer, and Cy5 was introduced at the5′-terminus. After completion of the synthesis, the oligonucleotide wasdeprotected and purified in a standard manner before use.[Oligonucleotide C (SEQ ID NO: 4)] gccaacaatg gaatgttgat tgggcccaaaccaccttcct ttcttgggat attggtccat gccaaaaggg agtattcgga gtcagtggaggogaaaaga<Composition of Hybridization Solution>

5×SSC (0.75 mol/L sodium chloride, 0.075 mol/l sodium citrate, pH 7.0)0.02% SDS (sodium lauryl sulfate)

The slice obtained in (6) and the above hybridization solution (1 ml)were poured into a HybriPack, followed by heat-sealing the top end ofthe pack. Hybridization was performed at 65° C. for 20 hours.

(8) Washing

The slice was removed from the HybriPack and washed under the conditionsshown in Table 2 in the order listed. The volume of a washing solutionwas 10 ml. TABLE 2 Composition of washing solution Washing temperatureWashing time   2 × SSC 0.2% SDS 25° C. 20 minutes 0.2 × SSC 0.2% SDS 25°C. 20 minutes 0.2 × SSC 0.2% SDS 55° C. 20 minutes 0.2 × SSC 0.2% SDS55° C. 20 minutes 0.2 × SSC 0.2% SDS 25° C. 20 minutes(9) Detection

The washed slice was placed on a non-fluorescent slide glass and a fewdrops of sterilized water were put onto the slice. The slide glass wasthen covered with a cover glass and mounted on a DNA chip detector(GeneTac V, Genomic Solutions K. K.), followed by detection using a Cy5laser. The image size was set to 10 μm per pixel.

(10) Fluorescence Intensity Measurement

The sum of fluorescence intensity obtained from 80 pixels around thecenter of each compartment was calculated as the intensity percompartment. FIG. 1 shows the fluorescence intensity obtained, alongwith an image of the washed hollow fiber and its surrounding area. Thecenter of each compartment was determined as appropriate. As a result,the distribution of fluorescence intensity in the hybridizedcompartments was uniform.

COMPARATIVE EXAMPLE 1

The same procedure as used in Example 1 was repeated, except thatmonomer solution A was replaced by monomer solution B.

[Monomer Solution B]

Acrylamide (0.475 g) and methylenebisacrylamide (0.025 g) were dissolvedin a 50/50 (by mass) mixture of glycerine and pure water to give a totalvolume of 10 ml.

FIG. 1 shows the fluorescence intensity obtained, along with an image ofthe washed hollow fiber and its surrounding area.

The distribution of fluorescence intensity in the hybridized hollowspace was uniform, but the fluorescence intensity decreased as comparedto Example 1.

COMPARATIVE EXAMPLE 2

The same procedure as used in Example 1 was repeated, except thatmonomer solution A was replaced by monomer solution C.

[Monomer Solution C]

Acrylamide (0.76 g) and methylenebisacrylamide (0.04 g) were dissolvedin a 50/50 (by mass) mixture of glycerine and pure water to give a totalvolume of 10 ml. FIG. 1 shows the fluorescence intensity obtained, alongwith an image of the washed hollow fiber and its surrounding area

The fluorescence intensity was lower than in Example 1, and thefluorescence intensity in the hollow space was high in the peripheralregion, but low in the center region.

EXAMPLE 2

The same procedure as used in Example 1 was repeated to prepare slices,except that monomer solution A and capture probe A were replaced bymonomer solution D and capture probe B (SEQ ID NO: 5), respectively.Capture probe B was constructed to have a terminal methacrylate group byintroducing an amino group at the 5′-terminus and then reacting the samewith glycidyl methacrylate.

[Monomer Solution D]

Dimethylacrylamide (0.27 g) and methylenebisacrylamide (0.03 g) weredissolved in a 50/50 (by mass) mixture of glycerine and pure water togive a total volume of 10 ml. [Capture probe B (SEQ ID NO: 5)]aaatacgcct gcaggcggag atcttccagg cccgcctcaa gggctggttc gagccaatagtggaagacat

Hybridization and washing were performed as follows.

(1) Hybridization

A hybridization solution was prepared, which was supplemented with 1pmol/ml oligonucleotide E (SEQ ID NO: 6) including, as a part thereof, acomplementary sequence to the nucleotide sequence of capture probe B(nucleotides 16 to 85 of SEQ ID NO: 6).

Oligonucleotide E was synthesized using an automated DNA synthesizer,and Cy5 was introduced at the 5′-terminus. After completion of thesynthesis, the oligonucleotide was deprotected and purified in astandard manner before use. [Oligonucleotide E (SEQ ID NO: 6)]gcccactggc gatgcatgtc ttccactatt ggctcgaacc agcccttgag gcgggcctggaagatctccg cctgcaggcg tatttgctgg gtctgttcc[Composition of Hybridization Solution]

6×SSC (0.75 mol/L sodium chloride, 0.075 mol/I sodium citrate, pH 7.0)0.02% SDS (sodium lauryl sulfate)

The resulting slice and the above hybridization solution (1 ml) werepoured into a HybriPack, followed by heat-sealing the top end of thepack. Hybridization was performed at 37° C. for 16 hours.

(2) Washing

The slice was removed from the HybriPack and washed under the conditionsshown in Table 3 in the order listed. The washing temperature was 45° C.The volume of a washing solution was 10 ml. TABLE 3 0.2 × SSC 0.1% SDS20 minutes 0.2 × SSC 0.1% SDS 20 minutes 0.2 × SSC 20 minutes(3) Detection

The washed slice was placed on a non-fluorescent slide glass and a fewdrops of sterilized water were put onto the slice. The slide glass wasthen covered with a cover glass and mounted on a DNA chip detector(GeneTac IV, Genomic Solutions K. K.), followed by detection using a Cy5laser. The image size was set to 10 μm per pixel.

(4) Fluorescence Intensity Measurement

The fluorescence intensity averaged over 200 pixels around the center ofeach compartment was calculated as the intensity per compartment.

FIG. 2 shows the fluorescence intensity obtained, along with an image ofthe washed hollow fiber and its surrounding area. The center of eachcompartment was determined as appropriate. As a result, the distributionof fluorescence intensity in the hybridized compartments was uniform.

EXAMPLE 3

The same procedure as used in Example 2 was repeated, except thatmonomer solution D was replaced by monomer solution A. FIG. 2 shows thefluorescence intensity per compartment, along with an image of thewashed hollow fiber and its surrounding area. The center of eachcompartment was determined as appropriate. As a result, the distributionof fluorescence intensity in the hybridized compartments was uniform.

COMPARATIVE EXAMPLE 3

The same procedure as used in Example 2 was repeated, except thatmonomer solution A was replaced by monomer solution E.

[Monomer Solution E]

N,N-Dimethylacrylamide (0.72 g) and methylenebisacrylamide (0.08 g) weredissolved in a 50/50 (by mass) mixture of glycerine and pure water togive a total volume of 10ml.

FIG. 2 shows the fluorescence intensity obtained, along with an image ofthe washed hollow fiber and its surrounding area. The fluorescenceintensity was lower than in Example 2, and the fluorescence intensity inthe hollow space was high in the peripheral region, but low in thecenter region.

COMPARATIVE EXAMPLE 4

The same procedure as used in Example 2 was repeated, except thatmonomer solution A was replaced by monomer solution F.

[Monomer Solution F]

N,N-Dimethylacrylamide (0.18 g) and methylenebisacrylamide (0.02 g) weredissolved in a 50/50 (by mass) mixture of glycerine and pure water togive a total volume of 10 ml.

The sliced chip did not hold any gel and its hollow spaces were notfilled (FIG. 2).

INDUSTRIAL APPLICABILITY

The present invention provides a biological substance-immobilized gel.The gel of the present invention is useful for detection of genes suchas DNA because its use ensures uniform fluorescence intensity throughoutthe compartment and achieves higher hybridization efficiency.

Sequence Listing Free Text

-   SEQ ID NO: 1: synthetic DNA-   SEQ ID NO: 2: synthetic DNA-   SEQ ID NO: 3: synthetic DNA-   SEQ ID NO: 4: synthetic DNA-   SEQ ID NO: 5: synthetic DNA-   SEQ ID NO: 6: synthetic DNA

1. A biological substance-immobilized gel which comprises a gel containing 2%-7% by mass of N,N-dimethylacrylamide and a biological substance immobilized on and/or in the gel.
 2. A biological substance-immobilized gel which comprises a gel having the following composition and a biological substance immobilized on and/or in the gel: (a) N,N-dimethylacrylamide   2% to 7% by mass (b) cross-linking agent 0.1% to 1.5% by mass.


3. The biological substance-immobilized gel according to claim 1 or 2, wherein the biological substance is a nucleic acid.
 4. The biological substance-immobilized gel according to claim 2 or 3, wherein the cross-linking agent is a multifunctional monomer having at least two ethylenically unsaturated bonds.
 5. The biological substance-immobilized gel according to claim 4, wherein the cross-linking agent is methylenebisacrylamide.
 6. A method for preparing a biological substance-immobilized gel, which comprises immobilizing a biological substance on and/or in a gel containing 2%-7% by mass of N,N-dimethylacrylamide.
 7. The method according to claim 6, wherein the gel is obtained by reacting 2%-7% by mass of N,N-dimethylacrylamide in the presence of 0.1%-1.5% by mass of a cross-linking agent.
 8. A gel-filled hollow tube which comprises a hollow tube whose hollow space is filled with the biological substance-immobilized gel according to any one of claims 1 to
 5. 9. The gel-filled hollow tube according to claim 8, wherein the hollow tube is a hollow fiber.
 10. A method for manufacturing a biological substance-immobilized gel microarray, which comprises allowing a plurality of gel-filled hollow tubes according to claim 8 or 9 to be tied in a bundle and cutting the resulting tube bundle in a direction intersecting with the longitudinal direction of the tubes.
 11. A method for manufacturing a biological substance-immobilized gel microarray, which comprises the following steps: (1) allowing a plurality of hollow tubes to be tied in a bundle; (2) filling the biological substance-immobilized gel according to any one of claims 1 to 5 into the hollow space of each tube in the resulting tube bundle; and (3) cutting the tube bundle in a direction intersecting with the longitudinal direction of the tubes.
 12. A biological substance-immobilized gel microarray which comprises the biological substance-immobilized gel according to any one of claims 1 to 5, wherein the gel is arranged in multiple compartments.
 13. The biological substance-immobilized gel microarray according to claim 12, wherein the surface area of each compartment is 10⁻⁶ m² or less.
 14. The biological substance-immobilized gel microarray according to claim 12 or 13, wherein the compartments are formed by slots or through holes.
 15. A biological substance-immobilized gel microarray which is obtained by allowing a plurality of gel-filled hollow tubes according to claim 8 or 9 to be tied in a bundle and cutting the tube bundle in a direction intersecting with the longitudinal direction of the tubes.
 16. The biological substance-immobilized gel microarray according to claim 15, wherein the hollow tubes are hollow fibers.
 17. A method for detecting a target to be measured, which comprises reacting an analyte with the microarray according to any one of claims 12 to 16 and detecting the target in the analyte.
 18. The method according to claim 17, wherein the target to be measured is a nucleic acid.
 19. The method according to claim 18, wherein the nucleic acid is 100 nucleotides or less in length. 